N1-2-thiophene-2-ylethyl-N2-substituted biguanide derivate, preparation method thereof, and pharmaceutical composition containing the same as active ingredients

ABSTRACT

The present invention provides an N1-2-thiophen-2-ylethyl-N2-substituted biguanide derivative of formula (I) or a pharmaceutically acceptable salt thereof, a method for preparing same, and a pharmaceutical composition comprising same as an active ingredient. The inventive N1-2-thiophen-2-ylethyl-N2-substituted biguanide derivative exhibits improved blood glucose level- and lipid level-lowering effects even with a reduced dosage as compared to conventional drugs, and thus, it is useful for preventing or treating diabetes, metabolic syndromes such as insulin-independent diabetes, obesity and atherosclerosis, or a P53 gene defect-related cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a National Stage of International Application No.PCT/KR2009/005856 filed Oct. 13, 2009, claiming priority based on KoreanPatent Application No. 10-2008-100221, filed Oct. 13, 2008, the contentsof all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to N1-2-thiophen-2-ylethyl-N2-substitutedbiguanide derivatives which exhibit improved blood glucose level- andlipid level-lowering effects even with a reduced dosage as compared toconventional drugs, a method for preparing same, and a pharmaceuticalcomposition comprising same as an active ingredient.

BACKGROUND ART

Diabetes is a disease characterized by continuous high blood glucoselevels. The major conditions of diabetes are carbohydrate metabolismabnormality and lipid metabolism abnormality, and systemic complicationsthereof grow worse due to blood flow disturbance caused by high bloodglucose levels and due to a decrease in sugar utilization. Such diabeticconditions are caused by the deficiency of hormone insulin, whichregulates carbohydrate and lipid metabolisms, or insulin resistance.Diabetes caused by insulin resistance is called “type 2 diabetes”.

Type 2 diabetes is induced from a state in which insulin does notexhibit a function of transporting glucose into cells due to thedecreases of insulin receptors or due to the deficiency of signaltransduction pathways through insulin receptors, i.e., a condition inwhich the body becomes resistant to insulin secreted from the pancreas.In other words, Type 2 diabetes suffers from direct destruction of bloodvessels and severe metabolic syndromes due to hyperinsulinemia.

Many kinds of anti-diabetic drugs have been used to treat type 2diabetes. However, the drugs excluding biguanide metformin do not showsatisfactory effects on the prevention of important complicationsincluding visual loss, heart failure, stroke, renal failure, peripheralneuropathy, foot ulcer and the like, although they are somewhateffective in lowering blood glucose levels. For example,sulfonylurea-based drugs, which act to lower blood glucose levels byenforcing insulin to be secreted from the pancreas, have problems inthat their effects rapidly disappear and they induce abnormal lipidmetabolism, thereby promoting arteriosclerosis, increasing bodyweightand causing brain injuries due to low blood glucose levels. Moreover, asglitazone-based drugs give an effect only on insulin resistance mainlyin adipose tissues, they must be used in combination with metformin. Inaddition, close attention should be paid to the use of theglitazone-based drugs because the drugs may cause side effects such asretinal vascular occlusion.

Metformin, the only drug that has the same effect as that of insulin,does not cause a low blood glucose problem, solves a problem of insulinresistance in adipose, liver and muscular tissues, and exhibits improvedblood glucose level- and glycosylated hemoglobin level-lowering actions.Particularly, it has been reported that metformin activates AMPK(AMP-activated protein kinase), and thus has various effects ofnormalizing blood glucose levels, enhancing lipid conditions,normalizing irregular menstruation, ovulation and pregnancy, treatingfatty liver, and even preventing cancers.

Metformin is generally administered three times a day, and its dosageper one administration is more than about 500 mg. Thus, in order to makesustained-release tablets to be administered once a day, tabletscontaining at least 1500 mg of metformin are required. However, it isdifficult to take such tablets due to its very large size. In addition,currently commercially available sustained-release tablets contain onlyabout 750 mg of metformin per one tablet, and thus, at least two tabletsshould be administered once at a 24 hr interval. Therefore, there existsa need to develop preparations which exhibit improved blood glucoselevel- and lipid level-lowering effects, thereby being capable ofreducing its daily dosage.

DISCLOSURE OF INVENTION

Accordingly, it is a primary object of the present invention to providea novel compound which exhibits improved blood glucose level- and lipidlevel-lowering effects even with a reduced dosage as compared toconventional drugs, and a method for preparing same.

It is another object of the present invention to provide apharmaceutical composition comprising said compound as an activeingredient, for preventing or treating diabetes, metabolic syndromessuch as insulin-independent diabetes, obesity and atherosclerosis, or aP53 gene defect-related cancer.

In accordance with one aspect of the present invention, there isprovided an N1-2-thiophen-2-ylethyl-N2-substituted biguanide derivativeof formula (I) or a pharmaceutically acceptable salt thereof:

wherein,

R is C₁-C₈alkyl; allyl; C₁-C₈alkoxyalkyl; C₃-C₇cycloalkyl;C₃-C₇cycloalkylC₁-C₃alkyl; substituted or non-substituted phenyl orphenylC₁-C₃alkyl; substituted or non-substituted naphthyl ornaphthylC₁-C₃alkyl; or adamantyl; and

R₁ is hydrogen or C₁-C₆alkyl.

In accordance with another aspect of the present invention, there isprovided a method for preparing the compound of formula (I).

In accordance with a further aspect of the present invention, there isprovided a pharmaceutical composition for preventing or treatingdiabetes, comprising the compound of formula (I) as an activeingredient.

In accordance with a further aspect of the present invention, there isprovided a pharmaceutical composition for preventing or treatingmetabolic syndromes, comprising the compound of formula (I) as an activeingredient.

In accordance with a further aspect of the present invention, there isprovided a pharmaceutical composition for preventing or treating a P53gene defect-related cancer, comprising the compound of formula (I) as anactive ingredient.

ADVANTAGEOUS EFFECTS

An N1-2-thiophen-2-ylethyl-N2-substituted biguanide derivative offormula (I) of the present invention is capable of exhibiting improvedblood glucose level- and lipid level-lowering effects even with areduced dosage, and thus, it is useful for preventing or treatingdiabetes, metabolic syndromes such as insulin-independent diabetes,obesity and atherosclerosis, or a P53 gene defect-related cancer.

BEST MODE FOR CARRYING OUT THE INVENTION

In the compound of formula (I) of the present invention, unlessotherwise stated or indicated, the term “C₁-C₈alkyl” used herein denoteseither a linear or branched alkyl group. Examples of said alkyl includemethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, n-hexyl, n-heptyl, and t-octyl.

Preferred examples of the term “C₁-C₈alkoxyalkyl” used herein includemethoxymethyl, methoxyethyl, methoxyethoxyethoxyethyl, ethoxymethyl, andoctyloxymethyl.

Examples of the term “C₃-C₇cycloalkyl” used herein include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

Preferred examples of the term “C₃-C₇cycloalkylC₁-C₃alkyl” used hereininclude cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl,cyclohexylethyl, cyclopropylpropyl, cyclobutylpropyl, cyclopentylpropyl,and cyclohexylpropyl.

Preferred examples of the term “substituted or non-substituted phenyl orphenylC₁-C₃alkyl” include benzyl, phenyl, naphthyl, 1-phenylethyl,2-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, and 3-phenylpropyl, whichhave either substituent(s) or no substituent. The number of thesubstituent(s) may be one to six, preferably one to three, and two ormore substituents may be each other same or different. Thesubstituent(s) may combine to any one of chemically allowable positionsin phenyl, and examples thereof include halogen, hydroxy, nitro, cyano,C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆cycloalkyl, C₆-C₁₀aryl, C₆-C₁₀aryloxy,C₁-C₆alkoxy, C₁-C₆haloalkoxy, C₃-C₆cycloalkyloxy, C₁-C₇alkanoyl,carboxy, carbamoyl, alkylamino, C₂-C₇sulfonate, sulfonamide, andC₁-C₆alkylthio.

Preferred examples of the term “substituted or non-substituted naphthylor naphthylC₁-C₃alkyl” include 1-naphthylmethyl, 2-naphthylmethyl,1-naphthylethyl, and 2-naphthylethyl, which have either substituent(s)or no substituent, wherein the substituent is the same as that of“phenyl or phenylC₁-C₃alkyl” as described above.

Preferred examples of the term “C₁-C₆alkyl” include methyl, ethyl,propyl, butyl, pentyl, and hexyl.

Among the compounds of formula (I) of the present invention, preferredare those wherein R is ethyl, t-octyl, 1-naphthyl, hexyl, methyl,cyclohexyl, t-butyl, benzyl, 2-ethylphenyl, propyl,3-trifluoromethylphenyl, 4-methoxyphenyl, 2,5-dimethoxyphenyl,3,4,5-trimethoxyphenyl, 4-fluorophenyl, 4-methylphenyl, phenyl,cyclopentyl, cycloheptyl, pentyl, methoxyethyl, butyl, 3-methylphenyl,4-t-butylphenyl, 3-hexylphenyl, 4-methoxyphenyl, 2,5-dimethoxyphenyl,3,5-dimethoxyphenyl, 4-bromophenyl, 3-bromophenyl, 3-phenylphenyl,3-phenoxyphenyl, 4-bromobenzyl, 4-methoxybenzyl, 3,5-dichlorophenyl,4-methylbenzyl, isopropyl, isobutyl, cyclopropylmethyl, or allyl; and R₁is hydrogen, 3-methyl, or 5-ethyl.

The pharmaceutically acceptable salt of the inventive compound offormula (I) includes salts with organic acids (for example, formic acid,acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid,trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaricacid, succinic acid, succinic acid monoamide, glutamic acid, tartaricacid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbicacid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid,benzenesulfonic acid, p-toluenesulfonic acid, and methansulfonic acid)and salts with inorganic acids (for example, hydrochloric acid, bromicacid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, andboric acid). The above-mentioned acid addition salts are prepared by ageneral salt preparation method comprising: (a) mixing the compound offormula (I) directly with an acid; (b) dissolving one of them in asolvent or a water-containing solvent and mixing the solution with theother one; or (c) placing the compound of formula (I) in either asolvent or an acid in a water-containing solvent and mixing them.

If the compound of formula (I) has an acidic group, for example, acarboxy group and a sulfonic acid group, it becomes an amphoteric salt.Examples of such a salt may include alkali metal salts (for example,sodium and potassium salts), alkaline earth metal salts (for example,calcium and magnesium salts), salts with inorganic acids (for example,aluminum and ammonium salts), basic addition salts (for example, saltswith organic acids such as trimethylamine, triethylamine, pyridine,picoline, ethanolamine, diethanolamine, triethanolamine,dicyclohexylamine, and N,N′-dibenzylethylenediamine), and a mixturethereof. Also, salts of the compound of formula (I) may be salts withbasic amino acids (for example, arginine, lysine and ornithine), or withacidic amino acids (for example, aspartic acid). The salt of thecompound of formula (I) is preferably a pharmaceutically acceptablesalt, more preferably an acid addition salt, even more preferablyacetate, hydrochloride, hydrobromide, methane sulfonate, malonate, oroxalate.

In one embodiment, the inventive compound of formula (I) can be preparedby:

1) subjecting a compound of formula (II) to a reaction with NaBH₄ in asolvent to form a compound of formula (III);

2) subjecting the compound of formula (III) to a reaction with thionylchloride in an organic solvent to form a compound of formula (IV);

3) subjecting the compound of formula (IV) to a reaction with sodiumcyanide in DMSO to form a compound of formula (V);

4) subjecting the compound of formula (V) to a reaction with sodiumborohydride and a nickel chloride hydrate in a solvent to form acompound of formula (VI);

5) subjecting the compound of formula (VI) to a reaction withR-isothiocyanate (RNCS) in an organic solvent in the presence of a baseto form a compound of formula (VIII); and

6) allowing the compound of formula (VIII) to be kept in a guanidinesolution in the presence of mercuric oxide:

wherein, R and R₁ have the same meanings as defined in formula (I).

In another embodiment, the inventive compound of formula (I) can beprepared by:

1) subjecting a compound of formula (II) to a reaction with NaBH₄ in asolvent to form a compound of formula (III);

2) subjecting the compound of formula (III) to a reaction with thionylchloride in an organic solvent to form a compound of formula (IV);

3) subjecting the compound of formula (IV) to a reaction with sodiumcyanide in DMSO to form a compound of formula (V);

4) subjecting the compound of formula (V) to a reaction with sodiumborohydride and a nickel chloride hydrate in a solvent to form acompound of formula (VI);

5) subjecting the compound of formula (VI) to a reaction with ethylchloroformate in an organic solvent in the presence of carbon disulfideand a base to form a compound of formula (VII);

6) subjecting the compound of formula (VII) to a reaction with NH₂R in asolvent in the presence of a base to form a compound of formula (VIII);and

7) allowing the compound of formula (VIII) to be kept in a guanidinesolution in the presence of mercuric oxide:

wherein, R and R₁ have the same meanings as defined in formula (I).

The above preparation methods are shown in Reaction Scheme 1 andprocedures thereof are described in detail as follows:

wherein, R and R₁ have the same meaning as defined in formula (I).

In step (1) of Reaction Scheme 1, the substituted thiophenecarboxaldehyde compound of formula (II) as a starting material issubjected to a reaction with sodium borohydride (NaBH₄) in a solvent(for example, methanol, ethanol, propanol, isopropanol, butanol,tetrahydrofuran, acetonitrile, N,N-dimethylformamide ordimethylsulfoxide) at a temperature ranging from 0° C. to a refluxtemperature to obtain the alcohol derivative of formula (III). Thesodium borohydride is used in an amount ranging from 1 to 3 moleequivalents based on the amount of the compound of formula (II).

The compound of formula (II) used as a starting material in step (1) iscommercially available and can be easily synthesized by a conventionalmethod.

In step (2) of Reaction Scheme 1, the compound of formula (III) obtainedin step (1) is subjected to a reaction with thionyl chloride in anorganic solvent (for example, dichloromethane, dichloroethane orN,N-dimethylformamide) at a temperature ranging from 0° C. to a refluxtemperature to obtain the chloride derivative of formula (IV). Thethionyl chloride is used in an amount ranging from 3 to 10 moleequivalents based on the amount of the compound of formula (III).

In step (3) of Reaction Scheme 1, the compound of formula (IV) obtainedin step (2) is subjected to a reaction with sodium cyanide in DMSO atroom temperature to obtain the cyanide derivative of formula (V). Thesodium cyanide is used in an amount ranging from 2 to 4 mole equivalentsbased on the amount of the compound of formula (IV).

In step (4) of Reaction Scheme 1, the compound of formula (V) obtainedin step (3) is subjected to a reaction with sodium borohydride and anickel chloride hydrate in a solvent (for example, methanol, ethanol,propanol, isopropanol, butanol, tetrahydrofuran, acetonitrile,N,N-dimethylformamide or dimethylsulfoxide) at a room temperature toobtain the substituted thiophene ethylamine derivative of formula (VI).The sodium borohydride and a nickel chloride hydrate may be used inamounts ranging from about 3 to 5 mole equivalents and from 1 to 2 moleequivalents, respectively, based on the amount of the compound offormula (V).

Further, the thiourea compound of formula (VIII) which is used as anintermediate in the synthesis of the compound of formula (I) is obtainedby: (A) in case isothiocyanate (RNCS) having a substituent R iscommercially available, subjecting the thiophene ethylamine compound offormula (VI) to a reaction with RNCS in a solvent in the presence of abase (step (1)); or (B) in case RNCS having a substituent R is notcommercially available, subjecting the compound of formula (VI) to areaction with ethyl chloroformate in a solvent in the presence of carbondisulfide and a base to form the compound of formula (VII) (step (1′)),and subjecting the compound of formula (VII) to a reaction with NH₂R inan organic solvent in the presence of a base (step (2)).

Examples of the base which may be used in steps (5), (5′) and (6) forthe synthesis of the thiophene ethylamine compound of formula (VI)include triethylamine, trimethylamine, diisopropylethylamine, and amixture thereof; and examples of the organic solvent includedichloromethane, dichloroethane, N,N-dimethylformamide, and a mixturethereof. The reactions in steps (5), (5′) and (6) may be conducted at atemperature ranging from 0° C. to room temperature. In steps (5) and(5′), the carbon disulfide, base and ethyl chloroformate may be used inamounts ranging from about 1 to 2 mole equivalents, respectively, basedon the amount of the compound of formula (VI).

In step (7) of Reaction Scheme 1, the thiourea compound of formula(VIII) obtained in step (2) is dissolved in a mixture of mercuric oxideand an appropriate organic solvent (e.g., ethylalcohol, methylalcohol orN,N-dimethylformamide), and 1M guanidine ethanol solution is added tothe resulting solution, which is refluxed. The mercuric oxide and 1Mguanidine ethanol solution may be used in amounts ranging from about 1to 2 mole equivalents and from 1 to 3 mole equivalents, respectively,based on the amount of the compound of formula (VIII). The reaction instep (3) may be conducted at a temperature ranging from room temperatureto the reflux temperature of the solvent used. For instance, in case ofusing a N,N-dimethylformamide solvent, the reaction temperature is inthe range of room temperature and 100° C. After completion of thereaction, the resulting solution is subjected to filtration, and then pHof the filtrate is preferably adjusted to the range of about 4 to 5 byusing an acid such as hydrochloric acid. The resulting solution isconcentrated and purified to obtain the compound of formula (I) or apharmaceutically acceptable salt thereof.

The compound of formula (I) or a pharmaceutically acceptable saltthereof thus obtained exhibits improved blood glucose level- and lipidlevel-lowering effects even with a reduced dosage as compared toconventional drugs, and therefore, it is useful for preventing ortreating metabolic syndromes.

Accordingly, the present invention provides a pharmaceutical compositionfor preventing or treating metabolic syndromes, comprising the compoundof formula (I) or a pharmaceutically acceptable salt thereof as anactive ingredient. Examples of the metabolic syndrome includeinsulin-independent diabetes, obesity and atherosclerosis.

In addition, the present invention provides a pharmaceutical compositionfor preventing or treating diabetes, comprising the compound of formula(I) or a pharmaceutically acceptable salt thereof as an activeingredient.

Furthermore, the present invention provides a pharmaceutical compositionfor preventing or treating a P53 gene defect-related cancer, comprisingthe compound of formula (I) or a pharmaceutically acceptable saltthereof as an active ingredient.

The compound of formula (I) or its pharmaceutically acceptable salt maybe administered as it is or in a form of a pharmaceutical composition incombination with at least one pharmaceutically acceptable additive.

The pharmaceutically acceptable additive may be selected inconsideration of the administration route and formulation purpose fromthe group consisting of stabilizing agents, binders, bases,sugar-containing coating agents, excipients, disintegrants,dissolution-serving agents, viscosity-controlling agents, coatingagents, emulsifiers, pH controlling agents, isotonic agents, polishingagents, dispersing agents, sweeteners, taste-enhancing agents, wettingagents, soft capsule bases, hard capsule bases, plasticizers,preservatives, anti-oxidants, solvents, lubricants, adhesives,surfactants, shielding agents, coloring agents, dispersing agents,topical analgesia, buffers, refreshing agents, dissolving agents, and amixture thereof.

The inventive pharmaceutical composition may be formulated in a formsuitable for a desired use and purpose by using a conventionalformulation technique.

The inventive pharmaceutical composition may be orally administered inthe form of tablets, powders, granules, pills, troches, capsules orsolutions. Also, the pharmaceutical composition may be parenterallyadministered in the form of injection solutions, suppositories,ointments, topical solutions, pastes, cataplasmas, liniments orplasters.

The suitable daily single dosage for oral administration of theinventive compound of formula (I) or a pharmaceutically acceptable saltthereof is preferably in the range of 0.5 and 150 mg/kg body weightbased on a 60 kg adult man. Such a dosage can be determined depending onvarious factors including the age and condition of the patient, and thechosen route of administration. In certain cases, either amount lessthan or greater than the above dosage can be administered.

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

Example 1 Preparation of N1-(thiophen-2-ylethyl)-N2-ethyl biguanidehydrochloride (1-1) Preparation of1-ethyl-3-(2-(thiophen-2-yl)ethyl)thiourea

Ethyl isothiocyanate (1.3 g, 15.0 mmol) was slowly added dropwise to asolution of thiopheneethylamine (1.3 g, 10.2 mmol) in dichloromethane(150 mL). Triethyl amine (3.8 mL, 20.4 mmol) was added dropwise to themixture and stirred at room temperature for 2 h. After completion of thereaction, pH of the resulting solution was adjusted to about 7 usingaqueous 1N HCl, to which water was added. The resulting mixture wasextracted with dichloromethane. The residue was purified by flash columnchromatography (ethyl acetate:hexanes=1:3), to give the title compound(1.9 g, 89%).

¹H NMR (300 MHz, CDCl₃) δ 7.45 (br s, 1H), 7.42 (br s, 1H), 7.39-7.30(m, 1H), 6.95-6.97 (m, 1H), 6.87-6.88 (m, 1H), 3.60-3.50 (m, 2H),3.40-3.25 (m, 2H), 3.00 (t, 2H, J=7.4), 1.02 (t, 3H, J=7.2)

(1-2) Preparation of N1-(thiophen-2-ylethyl)-N2-ethyl biguanidehydrochloride

The compound obtained in step (1-1) (1.1 g, 5.10 mmol) was dissolved inethanol (15 mL), and mercuric(II)oxide (2.2 g, 10.2 mmol) was addedthereto. Guanidine (15 mL, 1.0 M in EtOH, 15.3 mmol) was slowly addeddropwise to the reaction mixture and refluxed for 12-24 h. The resultingmixture was cooled and filtered with a Celite filter. pH of the filtratewas adjusted to about 4 to 5 using aqueous 2N HCl. The resulting mixturewas concentrated and the residue was purified by flash columnchromatography (dichloromethane:methanol=9:1), to obtain the titlecompound (0.21 g, 15%).

¹H NMR (300 MHz, DMSO-d₆) δ 7.33-7.34 (m, 1H), 6.85-6.94 (m, 2H),3.28-3.32 (m, 2H), 2.96-3.11 (m, 4H), 0.99-1.04 (m, 3H); mp 128.0-131.7°C.

Example 2 Preparation of N1-2-(thiophen-2-ylethyl)-N2-t-octyl biguanidehydrochloride (2-1) Preparation of 2-(2-isothiocyanatoethyl)thiophene

Thiopene ethylamine (4.60 mL, 39.31 mmol) and triethyl amine (5.00 mL,39.31 mmol) were dissolved in 1,2-dichloroethane (25 mL), and theresulting mixture was cooled to 0° C. A solution of carbon disulfide(2.36 mL, 39.31 mmol) in 1,2-dichloroethane (50 mL) was added dropwisethereto over 15 min. The reaction mixture was allowed to warm to roomtemperature and re-cooled to 0° C., to which ethyl chloroformate (4.27mL, 31.39 mmol) was added dropwise. The reaction mixture was stirred atroom temperature for about 1-2 h, and water (150 mL) and aqueous 2Nsodium hydroxide (75 mL) were sequentially added thereto. The resultingmixture was extracted with dichloromethane. The extract was washed withwater and brine, dried over sodium sulfate, and concentrated, to obtainthe yellow title compound in a liquid form (5.37 g, 80.75%).

1H NMR (300 MHz, CDCl₃) δ 7.22 (dd, 1H, J=5.1, 1.2 Hz, Thiopene), 6.98(dd, 1H, J=5.1, 3.4 Hz, Thiopene), 6.93-6.91 (m, 1H, Thiopene), 3.76 (t,2H, J=6.7 Hz, CH₂), 3.22 (t, 2H, J=6.8 Hz, CH₂)

(2-2) Preparation of 1-t-octyl-3-(2-(thiophen-2-yl)ethyl)thiourea

The procedure of step (1-1) of Example 1 was repeated except for usingthe compound obtained in step (2-1) (2.5 g, 15.0 mmol) and t-octyl amine(1.6 mL, 10.2 mmol), to obtain the title compound (2.5 g, 83%).

1H NMR (300 MHz, DMSO-d₆) δ 7.33 (m, 1H), 7.31 (br s, 1H), 7.19 (br s,1H), 6.93 (m, 1H), 6.84 (m, 1H), 3.57 (m, 2H), 2.96 (t, 2H, J=7.0), 1.97(s, 2H), 1.97 (s, 6H), 0.92 (s, 9H),

(2-3) Preparation of N1-2-(thiophen-2-ylehtyl)-N2-t-octyl biguanidehydrochloride

The procedure of step (1-2) of Example 1 was repeated except for usingthe compound obtained in step (2-2) (1.5 g, 5.10 mmol), to obtain thetitle compound (0.31 g, 17%).

¹H NMR (300 MHz, DMSO-d₆) δ 7.33-7.36 (m, 1H), 6.89-6.96 (m, 2H),3.20-3.21 (m, 2H), 2.97-2.99 (m, 2H), 1.28-1.30 (m, 6H), 0.90-0.93 (m,1H)

EXAMPLES 3 TO 44

The procedure of Example 1 was repeated except for using anisothiocyanate compound, which corresponds to a desired compound,instead of ethyl isothiocyanate in step (1-1) of Example 1, to obtainthe title compounds of Examples 3-5, 7, 8, 10-13, 20, 23, 25, 26, 41 and42.

Alternatively, the procedure of Example 2 was repeated except for usingan amine compound which corresponds to a desired compound, instead oft-octyl amine, in step (2-2) of Example 2, to obtain the title compoundsof Examples 6, 9, 14-19, 21, 22, 24, 27-40, 43 and 44.

Example 3 Preparation of N1-2-(thiophen-2-ylethyl)-N2-1-naphthylbiguanide hydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.71-7.72 (m, 1H), 7.28-7.50 (m, 5H),6.79-6.90 (m, 4H), 3.3.37-3.33 (m, 2H), 2.97-3.03 (t, 2H, J=6.9).

Example 4 Preparation of N1-2-(thiophen-2-ylethyl)-N2-hexyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.25-7.35 (m, 1H), 6.81-6.88 (m, 2H),3.20-3.24 (m. 2H), 2.88-3.05 (m, 4H), 2.89-2.99 (m, 4H), 1.11-1.32 (m,4H), 0.75-0.78 (m, 3H).

Example 5 Preparation of N1-2-(thiophen-2-ylethyl)-N2-methyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.31-7.32 (m, 1H), 6.87-6.95 (m, 2H),3.27-3.32 (t, 2H, J=7.2), 2.93-2.98 (t, 2H, J=7.2), 2.63 (s, 3H); Mass(ESI) m/z 226.7 (M⁺).

Example 6 Preparation of N1-2-(thiophen-2-ylethyl)-N2-cyclohexylbiguanide hydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.36-7.39 (m, 1H), 6.90-7.00 (m, 2H),3.25-3.24 (m, 2H), 2.97-3.05 (m, 2H), 2.23-2.28 (m, 1H), 1.66-1.90 (m,5H), 1.14-1.18 (m, 5H); Mass (ESI) m/z 294.7 (M⁺).

Example 7 Preparation of N1-2-(thiophen-2-ylethyl)-N2-t-butyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.38-7.42 (m, 1H), 6.94-7.01 (m, 2H),3.25-3.24 (m, 2H), 2.99-3.04 (m, 2H), 1.25-1.30 (m, 9H); Mass (ESI) m/z268.7 (M⁺).

Example 8 Preparation of N1-2-(thiophen-2-ylethyl)-N2-t-benzyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.24-7.48 (m, 6H), 6.90-7.01 (m, 2H),4.34-4.35 (m, 2H), 2.98-3.22 (m, 4H); Mass (ESI) m/z 302.7 (M⁺).

Example 9 Preparation of N1-2-(thiophen-2-ylethyl)-N2-2-ethylphenylbiguanide hydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.23-7.38 (m, 6H), 6.90-6.91 (m, 2H),3.27-3.39 (m, 4H), 2.98-3.00 (m, 2H), 2.77-2.79 (m, 2H); Mass (ESI) m/z316.5 (M⁺).

Example 10 Preparation of N1-2-(thiophen-2-ylethyl)-N2-methyl biguanidehydrochloride (isomer)

¹H NMR (300 MHz, DMSO-d₆) δ 7.34-7.36 (m, 1H), 6.92-6.95 (m, 2H),3.55-3.59 (t, 2H, J=7.4), 3.27-3.28 (m, 3H), 3.06-3.11 (t, 2H, J=7.4);mp 185.0-199.5° C.

Example 11 Preparation of N1-2-(thiophen-2-ylethyl)-N2-methyl biguanidehydrochloride (isomer)

¹H NMR (300 MHz, DMSO-d₆) δ 7.32-7.35 (m, 1H), 6.90-6.93 (m, 2H),3.28-3.53 (m, 2H), 2.95-3.16 (m, 2H), 2.80 (s, 3H).

Example 12 Preparation of N1-2-(thiophen-2-ylethyl)-N2-methyl biguanidehydrochloride (isomer)

¹H NMR (300 MHz, DMSO-d₆) δ 7.32-7.33 (m, 1H), 6.88-6.91 (m, 2H),3.47-3.51 (m, 2H), 3.01-3.05 (m, 2H), 2.90 (s, 3H).

Example 13 Preparation of N1-2-(thiophen-2-ylethyl)-N2-propyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.30-7.40 (m, 1H), 6.89-6.83 (m, 2H),3.34-3.45 (m, 2H), 2.93-3.01 (m, 4H), 1.41-1.50 (m, 2H), 0.78-0.85 (m,3H).

Example 14 Preparation ofN1-2-(thiophen-2-ylethyl)-N2-(3-trifluoromethyl)phenyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.47-7.22 (m, 5H), 6.92-6.86 (m, 2H),3.38-3.40 (m, 2H), 2.97-3.02 (m, 2H); mp 129-131° C.

Example 15 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-methoxy)phenylbiguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.26-7.28 (m, 1H), 7.02-7.05 (m, 2H), 6.85-6.92(m, 4H), 3.70 (s, 3H), 3.32-3.37 (m, 2H), 2.98-3.10 (m, 2H); mp 127-129°C.

Example 16 Preparation ofN1-2-(thiophen-2-ylethyl)-N2-(2,5-dimethoxy)phenyl biguanidehydrochloride

¹H NMR (300 MHz, D₂O) δ 7.26-7.29 (m, 1H), 6.83-6.94 (m, 3H), 6.67-6.70(m, 2H), 3.62 (s, 3H), 3.68 (s, 3H), 3.33 (t, 2H, J=7.0), 2.98 (t, 2H,J=7.1); mp 179-181° C.

Example 17 Preparation ofN1-2-(thiophen-2-ylethyl)-N2-(3,4,5-trimethoxy)phenyl biguanidehydrochloride

¹H NMR (300 MHz, D₂O) δ 7.25-7.23 (m, 2H), 6.91-6.85 (m, 3H), 3.65 (s,6H), 3.55 (s, 3H), 3.36 (t, 2H, J=7.0 Hz), 2.97 (t, 2H, J=7.3 Hz); mp92-94° C.

Example 18 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-fluorophenyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.29-7.31 (m, 1H, J=4.5 Hz), 7.14-7.11 (m, 4H),6.94-6.89 (m, 2H), 3.34 (t, 2H, J=7.5 Hz), 3.00 (t, 2H, J=7.3 Hz); mp83-85° C.

Example 19 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-methylphenyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.28-7.31 (m, 1H), 7.13-7.16 (m, 2H), 6.88-6.96(m, 4H), 3.38 (t, 2H, J=7.2 Hz), 3.01 (t, 2H, J=7.2 Hz), 2.21 (s, 3H);mp 97-99° C.

Example 20 Preparation of N1-2-(thiophen-2-ylethyl)-N2-phenyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.30-7.24 (m, 3H), 7.10-7.07 (m, 3H),6.94-6.88 (m, 2H), 3.38 (t, 2H, J=7.3 Hz), 3.01 (t, 2H, J=7.2 Hz).

Example 21 Preparation of N1-2-(thiophen-2-ylethyl)-N2-cyclopentylbiguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.27-7.26 (m, 1H), 6.92-6.90 (m, 1H), 6.85-6.83(m, 1H), 3.75-3.79 (m, 1H), 3.28 (t, 2H, J=7.2 Hz), 2.94 (t, 2H, J=7.2Hz), 1.82-1.73 (m, 2H), 1.60-1.53 (m, 2H), 1.49-1.33 (m, 4H).

Example 22 Preparation of N1-2-(thiophen-2-ylethyl)-N2-cycloheptylbiguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.28-7.30 (m, 1H), 6.86-6.94 (m, 2H), 3.48-3.51(m, 1H), 3.28-3.31 (m, 2H), 2.98-3.05 (m, 2H), 1.54-1.60 (m, 2H),1.13-1.45 (m, 10H).

Example 23 Preparation of N1-2-(thiophen-2-ylethyl)-N2-pentyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.33 (m, 1H), 6.90-6.95 (m, 2H), 3.49-3.55(m, 2H), 3.21-3.40 (m, 2H), 3.00-3.15 (m, 2H), 1.22-1.50 (m, 6H),0.79-0.87 (m, 3H).

Example 24 Preparation of N1-2-(thiophen-2-ylethyl)-N2-methoxyethylbiguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.32-7.33 (m, 1H), 6.93-6.95 (m, 1H), 6.86-6.88(m, 1H), 3.36-3.33 (m, 4H), 3.21 (s, 5H), 2.97-3.02 (m, 2H).

Example 25 Preparation of N1-2-(thiophen-2-ylethyl)-N2-butyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.30 (m, 1H), 6.93-6.88 (m, 2H), 3.50 (m,2H), 2.79-3.10 (m, 4H), 1.16-1.45 (m, 4H), 0.82-0.86 (m, 3H).

Example 26 Preparation of N1-2-(thiophen-2-ylethyl)-N2-butyl biguanidehydrochloride (isomer)

¹H NMR (300 MHz, DMSO-d₆) δ 7.19-7.30 (m 1H), 6.93-6.88 (m, 2H),3.30-3.40 (m, 2H), 2.70-3.29 (m, 4H), 1.15-1.60 (m, 4H), 0.83-0.88 (m,3H).

Example 27 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(3-methylphenyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.29-7.31 (m, 1H), 7.13-7.16 (m, 2H), 6.88-6.95(m, 4H), 3.37-3.41 (m, 2H), 2.99-3.03 (m, 2H), 2.21 (s, 3H).

Example 28 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-t-butylphenyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.27-7.30 (m, 3H), 7.01-7.04 (d, 2H, J=6.9),6.87-6.94 (m, 2H), 3.36-3.40 (m, 2H), 2.98-3.03 (m, 2H), 1.14 (s, 9H);mp 215.2-224.1° C.

Example 29 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-hexylphenyl)biguanide hydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.34-7.36 (m, 1H), 7.04-7.11 (m, 4H),6.87-6.97 (m, 2H), 3.32-3.43 (m, 2H), 2.91-3.04 (m, 2H), 2.47-2.50 (m,2H) 1.49-1.51 (m, 2H), 1.25-1.35 (m, 6H), 0.81-0.85 (s, 3H); mp155.8-160.8° C.

Example 30 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-methoxyphenyl)biguanide hydrochloride (isomer)

¹H NMR (300 MHz, D₂O) δ 7.25-7.27 (m, 1H), 7.02-7.05 (m, 2H), 6.84-6.92(m, 4H), 3.66 (s, 3H), 3.36-3.40 (m, 2H), 2.97-3.01 (m, 2H); mp98.6-99.2° C.

Example 31 Preparation ofN1-2-(thiophen-2-ylethyl)-N2-(2,5-dimethoxyphenyl) biguanidehydrochloride

¹H NMR (300 MHz, D₂O) δ 7.33-7.34 (m, 1H), 6.91-6.97 (m, 4H), 6.65-6.70(m, 1H), 3.73 (s, 3H), 3.70 (s, 3H), 3.33-3.39 (t, 2H, J=6.9), 2.95-3.03(t, 2H, J=6.9).

Example 32 Preparation ofN1-2-(thiophen-2-ylethyl)-N2-(3,5-dimethoxyphenyl) biguanidehydrochloride

¹H NMR (300 MHz, D₂O) δ 7.28-7.30 (m, 1H), 6.88-6.95 (m, 2H), 6.21-6.29(m, 3H), 3.66 (s, 6H), 3.37-3.42 (t, 2H, J=6.9), 2.99-3.04 (t, 2H,J=6.9); mp 162.8-167.1° C.

Example 33 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-bromophenyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.39-7.45 (m, 2H), 7.24-7.27 (m, 1H), 7.02-7.05(m, 2H), 6.84-6.95 (m, 2H), 3.35-3.42 (m, 2H), 2.96-3.05 (m, 2H); mp126.8-133.7° C.

Example 34 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(3-bromophenyl)biguanide hydrochloride

¹NMR (300 MHz, D₂O) δ 7.20-7.33 (m, 4H), 7.08-7.11 (m, 1H), 6.85-6.95(m, 2H), 3.36-3.41 (m, 2H), 2.95-3.05 (m, 2H); mp 107.1-111.6° C.

Example 35 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-phenylphenyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.57-7.63 (m, 4H), 7.38-7.44 (m, 2H), 7.19-7.36(m, 4H), 6.83-6.96 (m, 2H), 3.38-3.45 (m, 2H), 2.90-3.07 (m, 2H); mp145.8-150.6° C.

Example 36 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-phenoxyphenyl)biguanide hydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.27-7.37 (m, 3H), 7.09-7.14 (m, 3H),6.88-6.94 (m, 6H), 3.38 (t, 2H, J=7.2 Hz), 3.01 (t, 2H, J=7.2 Hz); mp87.1-96.0° C.

Example 37 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-bromobenzyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.43-7.50 (m, 2H), 7.22-7.28 (m, 1H), 7.09-7.17(m, 2H), 6.75-6.93 (m, 2H), 4.23 (s, 2H), 3.29-3.33 (m, 2H), 2.93-3.03(m, 2H); mp 102.1-103.5° C.

Example 38 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-methoxybenzyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.24-7.26 (d, 1H, J=6.7 Hz), 7.11-7.16 (d, 2H,J=6.7 Hz), 6.80-6.91 (m, 4H), 4.18 (s, 2H), 3.66 (s, 3H), 3.28-3.33 (t,2H, J=7.2 Hz), 2.92-3.02 (t, 2H, J=7.2 Hz).

Example 39 Preparation ofN1-2-(thiophen-2-ylethyl)-N2-(3,5-dichlorophenyl) biguanidehydrochloride

¹H NMR (300 MHz, D₂O) δ 7.21-7.27 (m, 2H), 7.10 (s, 2H), 6.88-6.95 (m,2H), 3.34-3.40 (m, 2H), 3.03-3.08 (m, 2H).

Example 40 Preparation of N1-2-(thiophen-2-ylethyl)-N2-(4-methylbenzyl)biguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.24-7.27 (m, 1H), 7.079-7.09 (m, 3H), 6.81-6.91(m, 3H), 4.34-4.37 (m, 2H), 3.28-3.33 (m, 2H), 2.96-3.04 (m, 2H), 2.25(s, 3H).

Example 41 Preparation of N1-2-(thiophen-2-ylethyl)-N2-isopropylbiguanide hydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.34-7.36 (m, 1H), 6.90-6.99 (m, 2H),3.70-3.75 (m, 1H), 3.35-3.40 (m, 2H), 2.95-3.00 (m, 2H), 1.06-1.08 (m,6H).

Example 42 Preparation of -2-(thiophen-2-ylethyl)-N2-isobutyl biguanidehydrochloride

¹H NMR (300 MHz, D₂O) δ 7.30-7.32 (m, 1H), 6.90-6.95 (m, 2H), 3.34-3.40(m, 2H), 3.99-3.04 (m, 2H), 2.90-2.98 (m, 2H), 1.73-1.77 (m, 1H),0.81-0.99 (m, 6H).

Example 43 Preparation of N1-2-(thiophen-2-ylethyl)-N2-cyclopropylmethylbiguanide hydrochloride

¹H NMR (300 MHz, D₂O) δ 7.30-7.31 (m, 1H), 6.91-6.94 (m, 2H), 3.36-3.41(m, 2H), 3.05-2.97 (m, 4H), 0.94-0.97 (m, 1H), 0.46-0.43 (m, 2H),0.19-0.18 (m, 2H).

Example 44 Preparation of N1-2-(thiophen-2-ylethyl)-N2-allyl biguanidehydrochloride

¹H NMR (300 MHz, DMSO-d₆) δ 7.28-7.30 (m, 1H), 6.90-6.93 (m, 2H),5.73-5.80 (m, 1H), 5.10-5.20 (m, 2H), 3.77-3.78 (m, 2H), 3.39-3.43 (m,2H), 2.99-3.04 (m, 2H); mp 97-99° C.

Example 45 Preparation of N1-2-(3-methylthiophen-2-ylethyl)-N2-methylbiguanide hydrochloride (45-1) Preparation of(3-methylthiophen-2-yl)methanol (Compound 3)

3-Methyl-2-thiophen-carboxaldehyde (1.00 g, 7.93 mmol) was dissolved inmethanol (16 mL), and the reaction mixture was cooled to 0° C., and thensodium borohydride (0.60 g, 15.86 mmol) was added. The reaction mixturewas stirred at room temperature for about 30 min, and water (30 mL) wassequentially added to the mixture. The resulting mixture was extractedwith ethyl acetate. The extract was washed with saline, dried oversodium sulfate, and concentrated. The residue was purified by flashcolumn chromatography (ethyl acetate:hexanes=20:80), to obtain the titlecompound (0.89 g, 88%).

¹H NMR (300 MHz, CDCl₃) δ 7.17 (d, 1H, thiophene), 6.84 (d, 1H,thiophene), 4.76 (d, 2H, J=4.92 Hz, CH₂), 2.25 (s, 3H, CH₃), 1.66 (t,1H, J=5.43 Hz, OH); IR (neat): 3320, 3100, 3060, 2918, 2861, 1555, 1430,1383, 1368, 1353, 1300, 1231, 1171, 1032, 996, 932, 876, 832, 703, 663cm−1; MS (ESI+) m/z 128.19 [M+].

(45-2) Preparation of 2-(3-methylthiophen-2-yl) acetonitrile (CompoundV)

Thionyl chloride (0.85 mL, 11.70 mmol) was slowly added dropwise todichloromethane (5 mL) comprising (3-methylthiophen-2-yl)methanolobtained in step (45-1) (0.50 g 3.90 mmol). The reaction mixture wasstirred at room temperature for about 30 minutes, and concentrated undera reduced pressure to obtain chloride (compound IV). The concentrate wasdissolved in dimethyl sulfoxide (2 mL). Sodium cyanide (0.57 g, 11.70mmol) was added thereto and stirred at room temperature for 1 hr. Water(30 mL) was sequentially added to the reaction mixture, and theresulting solution was extracted with ethyl acetate. The organic layerwas washed with brine, dried over sodium sulfate, and concentrated. Theresidue was purified by flash column chromatography (ethylacetate:hexane=20:80), to obtain the title compound (0.39 g, 56%).

¹NMR (300 MHz, CDCl₃) δ 7.16 (d, 1H, thiophene), 6.84 (d, 1H,thiophene), 3.78 (s, 2H, CH₂), 2.24 (s, 3H, CH₃); IR (neat): 3098, 2917,2248, 1557, 1432, 1409, 1383, 1367, 1298, 1237, 1159, 1083, 1023, 952,915, 879, 855, 828, 738, 706, 664 cm⁻¹.

(45-3) Preparation of 2-(3-methylthiophen-2-yl) ethane amine (CompoundVI)

Nickel chloride (0.95 g, 0.73 mmol) was slowly added dropwise to thesolution of 2-(3-methylthiophen-2-yl) acetonitrile obtained in step(45-2) (0.10 g 0.73 mmol) in ethanol (3 mL). To a reaction mixture wasadded portion wise sodium borohydride (0.83 g, 2.19 mmol), and wasstirred at room temperature. Water (30 mL) was sequentially added to themixture, and the resulting mixture was extracted with ethyl acetate. Theorganic layer was washed with brine, dried over sodium sulfate, andconcentrated. The residue was purified by flash column chromatography(dichloro methane:methanol=8:2), to obtain the title compound (51 mg,50%).

¹H NMR (300 MHz, CDCl₃) δ 7.09 (d, 1H, thiophene), 6.80 (d, 1H,thiophene), 3.21 (s, 4H, 2×CH₂), 2.21 (s, 3H, CH₃).

(45-4) Preparation of N1-2-(3-methylthiophen-2-yl)-N2-methyl thiourea

The procedure of step (1-1) of Example 1 was repeated except for usingthe compound obtained in step (45-3) (1.4 g, 10.2 mmol) to obtain thetitle compound (1.9 g, 87%).

¹H NMR (300 MHz, CDCl₃) δ 7.06 (d, 1H, J=5.1 Hz), 6.81 (d, 1H, J=5.1Hz), 6.40 (br s, 1H), 6.00 (br s, 1H), 3.74 (m, 2H), 3.05 (m, 2H), 2.89(m, 3H), 2.20 (s, 3H).

(45-5) Preparation of N1-2-(3-methylthiophen-2-ylethyl)-N2-methylbiguanide hydrochloride

The procedure of step (1-2) of Example 1 was repeated except for usingthe compound obtained in step (45-4) (1.1 g, 5.10 mmol) to obtain thetitle compound (0.23 g, 16%).

¹H NMR (300 MHz, D₂O) δ 7.02 (d, 1H, J=5.1 Hz), 6.64 (d, 1H, J=5.1 Hz),3.04-3.06 (m, 2H), 2.72-2.74 (m, 2H), 2.48 (s, 3H), 1.93 (s, 3H).

Example 46 Preparation of N1-2-(5-ethylthiophen-2-ylethyl)-N2-methylbiguanide hydrochloride

The procedure of Example 1 was repeated except for using the compound,which corresponds to a desired compound, instead of3-methyl-2-thiophen-carboxaldehyde in step (45-1) of Example 45, toobtain the desired compound.

¹H NMR (300 MHz, D₂O) δ 6.40-6.43 (m, 2H), 3.07-3.09 (m, 2H), 2.67-2.69(m, 2H), 2.46-2.49 (m, 5H), 0.91-0.96 (m, 3H).

TEST EXAMPLES

The pharmacological activities of the compounds prepared in Examples 1to 46 were determined as described in the following Test Examples 1 to4. Several tests were conducted three times on each of five plates whichwere grouped by the compounds of Examples 1 to 9, Examples 10 to 12,Examples 13 to 22, Examples 23 to 39, and Examples 40 to 46, and averagevalues were calculated from results of the tests. 2 mM metformin wasused as a comparative compound for each of the plates (control group).In this case, even though metformin having the same concentration wasused, test average values obtained were more or less different from eachother due to different measuring environments among the plates.

The glucose absorption ability was measured using insulin and thecontrol group simultaneously, and values of more than 200% inconsideration of deviations on the basis of cell-based experiments aredetermined to have a glucose absorption ability.

Test Example 1 Measurement of Cholesterol Synthesis Inhibitory Ability

A cholesterol synthesis inhibitory ability, which is an importantfunction of AMPK (AMP-activated protein kinase), was measured usingHepG2 cells (ATCC: American Type Culture Collection) as a hepatic cellmodel according to the following procedure.

The hepatic cell model HepG2 cells were cultured in 1% serum-containingmedia (DMEM (Gibco), FBS (Gibco)) for 24 hours, and then treated with100 μM or 500 μM of each of the compounds prepared in Examples for 24hours. Then, the cells were disrupted with a lysis solution (0.1 Mpotassium phosphate, pH 7.4, 0.05 M NaCl, 5 mM cholic acid, 0.1% TritonX-100 (Sigma)). To the disrupted cells was added the same volume of areaction solution (2 U/mL of a cholesterol oxidizing agent, 2 U/mL of aperoxidase, 0.2 U/mL of a cholesterol esterase, and 300 μM of Amplex redas a fluorescent factor (Molecular Probe)), which was allowed to reactat 37° C. for 30 minutes. After completion of the reaction, the contentof cholesterol formed in the cell lysates was measured at a wavelengthof 560/590 nm (ex/em) using a fluorescence microscope.

A lower measurement value means an increased cholesterol synthesisinhibitory ability. If the inventive compounds (a 100 μM or 500 μMconcentration) show values less than the value of the control group (a 2mM concentration), it is understood that they are much superior to thecontrol group by comparison of the used amounts. For example, thecompound of Example 1 showed a lower value than the control group at aconcentration of 100 μM, suggesting that the effect in inhibitingsynthesis of the compound was better by at least 20 times than that ofthe control group.

Test Example 2 Measurement of Triglyceride

HepG2 cells (ATCC: American Type Culture Collection) were cultured in 1%serum-containing media (DMEM (Gibco), FBS (Gibco)) for 24 hours, andthen treated with 100 μM or 500 μM of each of the compounds prepared inExamples for 24 hours. Then, the cells were disrupted with a lysissolution (0.1 M potassium phosphate, pH 7.4, 0.05 M NaCl, 5 mM cholicacid, 0.1% Triton X-100). To the disrupted cells was added the samevolume of a reaction solution (0.76 U/mL of a glycerol kinase (AsanPharmaceutical Co., Ltd.), 151333 U/mL of a peroxidase (AsanPharmaceutical Co., Ltd.), 22.2 U/mL of a glycerol oxidizing agent (AsanPharmaceutical Co., Ltd.), and 300 μM of Amplex red as a fluorescentfactor (Molecular Probe)), which was allowed to react at 37° C. for 30minutes. After completion of the reaction, the content of triglycerideformed in the cell lysates was measured at a wavelength of 560/590 nm(ex/em) using a fluorescence microscope.

A lower measurement value means an increased triglyceride synthesisinhibitory ability. If the inventive compounds (a 100 μM or 500 μMconcentration) show values less than the value of the control group (a 2mM concentration), it is understood that they are much superior to thecontrol group by comparison of the used amounts. For example, thecompound of Example 1 showed a lower value than the control group at aconcentration of 100 μM, suggesting that the effect in inhibitingtriglyceride synthesis of the compound was better by at least 20 timesthan that of the control group.

Test Example 3 Measurement of Gluconeogenesis Inhibitory Ability

HepG2 cells (ATCC: American Type Culture Collection) were cultured in10% serum-containing high glucose media, transferred to noserum-containing low glucose media (DMEM (Gibco), FBS (Gibco)), and thentreated with 100 μM of each of the compounds for 24 hours. Then, thecells were treated with 0.5 μCi ¹⁴C-lactate (Amersham Pharmacia) and 10mM L-lactate (Sigma), and cultured for 4 hours. After cultivation, themedia were removed therefrom, and the cells were washed with PBS,treated with 0.1N NaOH and kept at room temperature for 1 hour. Then,the cells were neutralized with 1N HCl, and the content of glucoseformed in the cells was measured with a liquid scintillation counter.

A lower measurement value means an increased gluconeogenesis inhibitoryability. If the inventive compounds (a 100 μM concentration) show valuesless than the value of the control group (a 2 mM concentration), it isunderstood that they are much superior to the control group bycomparison of the used amounts. For example, the compound of Example 1showed a lower value than the control group at a concentration of 100μM, suggesting that the effect in inhibiting gluconeogenesis of thecompound was better by at least 20 times than that of the control group.

Test Example 4 Measurement of Glucose Absorption Ability

C2C12 cells (ATCC: American Type Culture Collection), a muscle cellmodel, were induced to differentiate into muscle cells in 2% bovinefetal serum-containing media (DMEM (Gibco), FBS (Gibco)) for 6 days. TheC2C12 cells which differentiated into muscle cells were transferred toserum-free low glucose media (DMEM (Gibco), FBS (Gibco)), treated with100 μM of each of the compounds, and then cultured with 1 μM of insulinfor 24 hours. After cultivation, the cells were treated with 1 μCi³H-deoxy-glucose (Amersham Pharmacia) and 10 μM deoxy-glucose (Sigma) at37° C. for 15 minutes. The media were removed therefrom, and the cellswere washed twice with PBS (phosphate buffered saline). The washed cellswere treated with 0.1N NaOH and neutralized with 1N HCl. The content ofglucose absorbed into the cells was measured with a liquid scintillationcounter.

A higher measurement value means a stronger ability to reduce insulinresistance. The compound of Example 1 showed a value similar to thecontrol group at a concentration of 100 μM, suggesting that the glucoseabsorption effect of the compound was better by about 20 times than thatof the control group.

TABLE 1 Gluconeogenesis Glucose Triglyceride Triglyceride CholesterolCholesterol inhibition absorption (100 uM) (500 uM) (100 uM) (500 uM)(100 uM) (100 uM) Ex. Aver. S.D. Aver. S.D. Aver. S.D. Aver. S.D. Aver.S.D. Aver. S.D. 1 56.70 4.08 54.57 8.62 45.18 8.80 30.51 11.3 48.92 4.03322.7 0.44 2 29.21 2.82 −1.92 3.58 63.63 25.3 55.38 8.48 68.28 23.8110.46 0.90 3 5.28 1.75 −6.37 0.19 30.90 0.99 31.14 9.71 3.73 0.49 15.102.52 4 22.80 2.09 3.22 5.88 33.81 3.85 51.74 9.38 26.40 3.71 14.31 1.415 65.03 3.08 41.59 3.62 49.29 4.16 41.36 3.08 58.50 9.23 350.4 8.20 651.00 6.37 −1.71 0.29 30.58 1.33 0.51 3.91 33.59 2.07 56.15 34.25 774.43 6.97 16.49 2.20 42.51 2.27 2.79 3.21 39.98 6.89 21.59 5.91 8 77.652.46 30.78 3.85 37.77 7.16 7.28 4.82 81.27 11.83 243.3 15.64 9 74.994.62 19.51 2.82 39.25 3.31 7.55 3.05 74.23 10.44 252.6 12.74Gluconeogenesis Glucose Triglyceride Cholesterol inhibition absorptionSt. St. St. St. Aver. Dev. Aver. Dev. Aver. Dev. Aver. Dev. Metformin69.58 4.70 74.50 3.05 59.14 4.44 335.45 3.50 (2 mM) GluconeogenesisGlucose Triglyceride Cholesterol Inhibition Absorption (100 uM) (100 uM)(100 μM) (100 μM) St. St. St. St. Ex. Aver. Dev. Aver. Dev. Aver. Dev.Aver. Dev. 10 76.16 3.12 52.29 4.14 5.72 6.45 6.91 0.42 11 74.98 1.7671.14 2.05 25.03 0.34 321.21 15.07 12 67.43 0.71 69.94 1.28 13.05 1.03221.61 3.53 Gluconeogenesis Glucose Triglyceride Cholesterol inhibitionabsorption St. St. St. St. Aver. Dev. Aver. Dev. Aver. Dev. Aver. Dev.Metformin 73.61 0.78 85.91 4.18 53.09 1.79 291.63 2.13 (2 mM)Gluconeogenesis Glucose Triglyceride Triglyceride CholesterolCholesterol inhibition absorption (100 uM) (500 uM) (100 uM) (500 uM)(100 uM) (100 uM) Ex. Aver. S.D. Aver. S.D. Aver. S.D. Aver. S.D. Aver.S.D. Aver. S.D. 13 59.3 3.19 48.9 2.26 77.1 0.94 61.7 4.54 73.51 1.85110.2 2.19 14 11.8 0.12 13.2 1.42 23.3 1.95 41.4 3.25 8.75 0.78 4.380.03 15 76.6 1.79 41.4 2.27 67.3 1.31 39.7 1.71 86.69 0.95 124.3 20.0 1673.8 1.78 51.3 5.56 85.0 2.80 58.5 1.08 68.89 0.59 147.3 15.0 17 86.23.11 79.4 0.93 87.2 1.21 57.5 0.57 95.56 0.44 165.2 24.1 18 67.0 0.7239.5 0.79 50.3 2.34 23.4 7.63 86.69 7.23 88.16 8.3 19 67.7 1.71 46.40.81 52.9 1.52 29.6 6.83 74.32 5.39 89.16 14.1 20 67.5 3.55 57.6 1.7045.7 3.64 18.3 0.50 87.36 0.54 125.4 6.1 21 80.5 8.51 85.6 12.7 68.24.05 45.8 2.55 97.66 1.70 174.8 36.1 22 77.8 10.8 78.9 3.55 69.8 2.9644.4 0.55 96.63 0.47 173.8 25.8 Gluconeogenesis Glucose TriglycerideCholesterol inhibition absorption St. St. St. St. Aver. Dev. Aver. Dev.Aver. Dev. Aver. Dev. Metformin 70.05 7.44 70.69 5.37 78.33 4.63 170.9434.76 (2 mM) Gluconeogenesis Glucose Triglyceride TriglycerideCholesterol Cholesterol inhibition absorption (100 uM) (500 uM) (100 uM)(500 uM) (100 uM) (100 uM) Ex. Aver. S.D. Aver. S.D. Aver. S.D. Aver.S.D. Aver. S.D. Aver. S.D. 23 13.83 1.36 17.16 4.48 11.05 7.53 15.661.52 2.96 0.23 0.00 0.00 24 77.91 2.45 71.93 4.01 92.58 3.64 75.75 5.2778.89 3.68 114.91 10.20 25 19.21 2.64 17.02 1.78 32.66 4.36 19.87 9.010.00 0.00 2.76 0.95 26 47.59 5.12 30.67 2.75 43.20 1.43 13.28 3.08 81.705.80 18.34 6.59 27 46.45 0.09 20.64 0.87 33.26 1.79 28.82 6.97 56.425.83 12.91 0.85 28 15.55 2.00 15.21 3.73 27.49 2.84 26.60 1.77 2.57 0.134.29 1.06 29 8.04 0.53 42.57 2.20 30.10 1.79 119.39 1.13 1.26 1.78 3.960.00 30 91.91 2.09 48.42 2.51 73.64 5.79 26.15 0.69 105.18 7.64 148.421.48 31 81.74 9.20 57.32 3.10 75.67 4.31 49.39 2.02 96.29 2.23 168.203.55 32 70.80 5.80 28.47 2.68 48.02 0.58 26.70 3.38 152.19 6.97 11.680.13 33 29.12 1.51 27.48 1.87 24.52 1.99 31.66 5.58 17.03 1.45 14.248.87 34 26.57 1.55 19.49 0.70 26.00 2.80 32.53 0.88 4.96 0.46 6.05 0.8135 18.94 0.81 14.92 0.26 28.27 2.31 30.49 0.22 109.62 6.94 35.00 3.02 3620.95 1.65 17.89 2.26 31.22 2.57 34.74 1.72 1.54 2.18 4.50 0.11 37 61.413.28 20.15 1.52 28.36 2.40 30.84 2.25 89.96 4.50 7.71 0.88 38 100.898.04 84.10 8.22 80.35 3.00 44.72 4.75 120.13 1.43 195.22 0.77 39 73.503.40 72.66 3.64 69.65 2.63 61.03 0.85 13.04 0.01 188.26 18.07Gluconeogenesis Glucose Triglyceride Cholesterol inhibition absorptionSt. St. St. St. Aver. Dev. Aver. Dev. Aver. Dev. Aver. Dev. Metformin74.57 3.40 73.31 1.95 64.50 2.23 253.20 9.49 (2 mM) GluconeogenesisGlucose Triglyceride Triglyceride Cholesterol Cholesterol inhibitionabsorption (100 uM) (500 uM) (100 uM) (500 uM) (100 uM) (100 uM) Ex.Aver. S.D. Aver. S.D. Aver. S.D. Aver. S.D. Aver. S.D. Aver. S.D. 4084.73 7.28 60.37 4.67 72.58 2.06 57.11 4.18 81.23 4.77 75.38 11.97 4193.91 8.33 81.62 7.49 87.73 6.99 75.54 1.34 97.81 5.57 150.65 27.61 4297.66 1.29 88.50 5.71 89.11 5.47 73.15 0.31 99.91 4.04 171.92 34.47 4390.52 2.75 79.92 2.45 88.47 5.68 78.80 2.40 99.28 8.86 196.03 2.15 4496.99 8.16 74.60 2.63 85.73 0.59 82.40 2.02 77.80 19.09 87.78 0.00 4592.38 6.36 89.00 0.63 77.03 1.82 63.78 2.57 94.51 8.75 264.76 42.77 4690.82 5.43 86.07 2.41 84.52 7.04 71.64 6.77 23.65 1.80 353.21 10.62Gluconeogenesis Glucose Triglyceride Cholesterol inhibition absorptionSt. St. St. St. Aver. Dev. Aver. Dev. Aver. Dev. Aver. Dev. Metformin81.14 3.17 65.31 17.17 39.79 0.83 365.22 23.75 (2 mM)

While the invention has been described with respect to the specificembodiments, it should be recognized that various modifications andchanges may be made by those skilled in the art to the invention whichalso fall within the scope of the invention as defined as the appendedclaims.

What is claimed is:
 1. An N1-2-thiophen-2-ylethyl-N2-substitutedbiguanide derivative of formula (I) or a pharmaceutically acceptablesalt thereof:

wherein, R is C₁-C₈alkyl; allyl; C₁-C₈alkoxyalkyl; C₃-C₇cycloalkyl;C₃-C₇cycloalkylC₁-C₃alkyl; substituted or non-substituted phenyl orphenylC₁-C₃alkyl; substituted or non-substituted naphthyl ornaphthylC₁-C₃alkyl; or adamantyl; and R₁ is hydrogen or C₁-C₆alkyl. 2.The compound or its pharmaceutically acceptable salt of claim 1, whereinR is selected from the group consisting of methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,n-heptyl, n-octyl, allyl, adamantyl, methoxymethyl, methoxyethyl,methoxyethoxyethoxyethyl, ethoxymethyl, octyloxymethyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl,cyclobutylethyl, cyclopentylethyl, cyclohexylethyl, cyclopropylpropyl,cyclobutylpropyl, cyclopentylpropyl, cyclohexylpropyl, benzyl, phenyl,naphthyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, 2-phenylpropyl,3-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl, 1-naphthylethyl, and2-naphthylethyl, wherein, benzyl, phenyl, naphthyl, 1-phenylethyl,2-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl,1-naphthylmethyl, 2-naphthylmethyl, 1-naphthylethyl, or 2-naphthylethyloptionally has one to six substituents, the substituents eachindependently being selected from the group consisting of halogen,hydroxy, nitro, cyano, C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆cycloalkyl,C₆-C₁₀aryl, C₆-C₁₀aryloxy, C₁-C₆alkoxy, C₁-C₆haloalkoxy,C₃-C₆cycloalkyloxy, C₁-C₇alkanoyl, carboxyl, carbamoyl, alkylamino,C₂-C₇sulfonate, sulfonamide, and C₁-C₆alkylthio; and R₁ is hydrogen,methyl, ethyl, propyl, butyl, pentyl, or hexyl.
 3. The compound or itspharmaceutically acceptable salt of claim 2, wherein R is ethyl,t-octyl, 1-naphthyl, hexyl, methyl, cyclohexyl, t-butyl, benzyl,2-ethylphenyl, propyl, 3-trifluoromethylphenyl, 4-methoxyphenyl,2,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 4-fluorophenyl,4-methylphenyl, phenyl, cyclopentyl, cycloheptyl, pentyl, methoxyethyl,butyl, 3-methylphenyl, 4-t-butylphenyl, 4-hexylphenyl, 4-methoxyphenyl,2,5-dimethoxyphenyl, 3,5-dimethoxyphenyl, 4-bromophenyl, 3-bromophenyl,4-phenylphenyl, 4-phenoxyphenyl, 4-bromobenzyl, 4-methoxybenzyl,3,5-dichlorophenyl, 4-methylbenzyl, isopropyl, isobutyl,cyclopropylmethyl, 2-(2-(2-methoxyethoxy)ethoxy)ethyl, or allyl; and R₁is hydrogen, 3-methyl, or 5-ethyl.
 4. The compound or itspharmaceutically acceptable salt of claim 1, wherein thepharmaceutically acceptable salt is a salt with an acid selected fromthe group consisting of formic acid, acetic acid, propionic acid, lacticacid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid,maleic acid, malonic acid, fumaric acid, succinic acid, succinic acidmonoamide, glutamic acid, tartaric acid, oxalic acid, citric acid,glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalicacid, salicylic acid, anthranilic acid, benzenesulfonic acid,p-toluenesulfonic acid, methansulfonic acid, hydrochloric acid, bromicacid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, andboric acid.
 5. A method for preparing a compound of formula (I), whichcomprises the steps of: 1) subjecting a compound of formula (II) to areaction with NaBH₄ in a solvent to form a compound of formula (III); 2)subjecting the compound of formula (III) to a reaction with thionylchloride in an organic solvent to form a compound of formula (IV); 3)subjecting the compound of formula (IV) to a reaction with sodiumcyanide in dimethylsulfoxide to form a compound of formula (V); 4)subjecting the compound of formula (V) to a reaction with sodiumborohydride and a nickel chloride hydrate in a solvent to form acompound of formula (VI); 5) subjecting the compound of formula (VI) toa reaction with R-isothiocyanate in an organic solvent in the presenceof a base to form a compound of formula (VIII); and 6) allowing thecompound of formula (VIII) to be kept in a guanidine solution in thepresence of mercuric oxide:

wherein R is C₁-C₈alkyl; allyl; C₁-C₈alkoxyalkyl; C₃-C₇cycloalkyl;C₃-C₇cycloalkylC₁-C₃alkyl; substituted or non-substituted phenyl orphenylC₁-C₃alkyl; substituted or non-substituted naphthyl ornaphthylC₁-C₃alkyl; or adamantyl; and R₁ is hydrogen or C₁-C₆alkyl.
 6. Amethod for preparing a compound of formula (I), which comprises thesteps of: 1) subjecting a compound of formula (II) to a reaction withNaBH4 in a solvent to form a compound of formula (III); 2) subjectingthe compound of formula (III) to a reaction with thionyl chloride in anorganic solvent to form a compound of formula (IV); 3) subjecting thecompound of formula (IV) to a reaction with sodium cyanide indimethylsulfoxide to form a compound of formula (V); 4) subjecting thecompound of formula (V) to a reaction with sodium borohydride and anickel chloride hydrate in a solvent to form a compound of formula (VI);5) subjecting the compound of formula (VI) to a reaction with ethylchloroformate in an organic solvent in the presence of carbon disulfideand a base to form a compound of formula (VII); 6) subjecting thecompound of formula (VII) to a reaction with NH₂R in a solvent in thepresence of a base to form a compound of formula (VIII); and 7) allowingthe compound of formula (VIII) to be kept in a guanidine solution in thepresence of mercuric oxide:

wherein R is C₁-C₈alkyl; allyl; C₁-C₈alkoxyalkyl; C₃-C₇cycloalkyl;C₃-C₇cycloalkylC₁-C₃alkyl; substituted or non-substituted phenyl orphenylC₁-C₃alkyl; substituted or non-substituted naphthyl ornaphthylC₁-C₃alkyl; or adamantyl; and R₁ is hydrogen or C₁-C₆alkyl. 7.The method of claim 5, wherein the base is selected from the groupconsisting of triethylamine, trimethylamine, diisopropylethylamine, anda mixture thereof; and the organic solvent is selected from the groupconsisting of dichloromethane, dichloroethane, dimethylformamide, and amixture thereof.
 8. The method of claim 6, wherein the base is selectedfrom the group consisting of triethylamine, trimethylamine,diisopropylethylamine, and a mixture thereof; and the organic solvent isselected from the group consisting of dichloromethane, dichloroethane,dimethylformamide, and a mixture thereof.
 9. A pharmaceuticalcomposition, comprising the compound or its pharmaceutically acceptablesalt of claim 1 as an active ingredient.
 10. The pharmaceuticalcomposition of claim 9, wherein the pharmaceutically acceptable salt isa salt with an acid selected from the group consisting of formic acid,acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid,trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaricacid, succinic acid, succinic acid monoamide, glutamic acid, tartaricacid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbicacid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid,benzenesulfonic acid, p-toluenesulfonic acid, methansulfonic acid,hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitricacid, carbonic acid, and boric acid.
 11. A method for treating ametabolic syndrome, which comprises administering the compound or itspharmaceutically acceptable salt of claim 1 to a mammal in need thereof,wherein the metabolic syndrome is selected from the group consisting ofcholesterol synthesis, gluconeogenesis, glucose absorption, andtrigylceride synthesis.
 12. A method for treating type 2 diabetes, whichcomprises administering the compound or its pharmaceutically acceptablesalt of claim 1 to a mammal in need thereof.